Team:Tokyo-NoKoGen/Achievement

From 2012.igem.org

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<BR>Pconst.(High)-RBS-NpSRII-9a.a.linker_NpHtrII-EnvZ-Double terminator-PompR-RBS-GFP-Double terminator</b>
<BR>Pconst.(High)-RBS-NpSRII-9a.a.linker_NpHtrII-EnvZ-Double terminator-PompR-RBS-GFP-Double terminator</b>
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<BR>Tokyo-NoKoGen 2010 created a chimeric sensory rhodopsin with a chemotaxis transducer tar from <i>E. coli</i> (BBa_K225001). We created a blue light sensor, a chimeric sensor rhodopsin, the same sensor domain of rodopsin from <i>Natronobacterium pharaonis (N. pharaonis)</i>, and tested to see if the GFP under PompC will show any expression under the light, the light signal received by the sensory rhodopsin. The figure below shows the result. As you can see, we have found that
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contrary to what we expected, the blue light sensor responded to light by turning off the expression of GFP, and turns on GFP expression in the dark.
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<div align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png height=60% width=60%></div>
<div align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png height=60% width=60%></div>
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<div align=center><img src=https://static.igem.org/mediawiki/2012/6/67/Figure.e.jpg height=60% width=60%></div>
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<div align=center><img src=https://static.igem.org/mediawiki/2012/6/67/Figure.e.jpg height=60% width=70%></div>
<BR>Left:BBa_K769022  
<BR>Left:BBa_K769022  
<BR>Middle:BBa_K769011
<BR>Middle:BBa_K769011
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<BR>Promoter(Pbad)-RBS-lux operon-DT</b>
<BR>Promoter(Pbad)-RBS-lux operon-DT</b>
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<BR>we have cloned the lux operon Photobacterium phosphoreum and expressed it under arabinose inducible promoter, PBad. We have expressed it inside <i>E. coli</i> and evaluated the luminescence at different temperature, at 30 and 20 degrees celcius. As you can see from the graph below, the strains grown under 20 degrees celcius showed a 5 times higher luminescence intensity than those grown under 30 degrees.
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<div align=center><img src=https://static.igem.org/mediawiki/2012/thumb/4/43/%E5%9F%B9%E9%A4%8A%E6%9D%A1%E4%BB%B6%EF%BC%91.png/665px-%E5%9F%B9%E9%A4%8A%E6%9D%A1%E4%BB%B6%EF%BC%91.png height=60% width=60%></div>
<div align=center><img src=https://static.igem.org/mediawiki/2012/thumb/4/43/%E5%9F%B9%E9%A4%8A%E6%9D%A1%E4%BB%B6%EF%BC%91.png/665px-%E5%9F%B9%E9%A4%8A%E6%9D%A1%E4%BB%B6%EF%BC%91.png height=60% width=60%></div>
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<BR>lux operon(K769011) + lumP(K769019)</b>
<BR>lux operon(K769011) + lumP(K769019)</b>
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<BR>To obtain luminescence of a different color, we have aimed to use a protein called lumazine (<i>lumP</i>) to shift the wavelength peak of the lux luminescence more towards blue light. As you can see in the figure, the wavelength shifted from 487 to 478 nm.
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<div align=center><img src=https://static.igem.org/mediawiki/2012/1/10/Lux+lumP.jpg height=60% width=60%></div>
<div align=center><img src=https://static.igem.org/mediawiki/2012/1/10/Lux+lumP.jpg height=60% width=60%></div>
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<BR>lux operon(K769011) + GFP(I13522)</b>
<BR>lux operon(K769011) + GFP(I13522)</b>
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<BR>To obtain another color of a luminescence, we have aimed to use the phenomenon BRET, expressing the GFP together with lux operon, so the luminescence of the lux operon will excite the GFP. As a result, we obtained a wavelength shift in the construct expressing the two together.
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<div align=center>
<div align=center>

Latest revision as of 03:42, 27 September 2012

  • achievement

Achievement


We have successfully managed to accomplish the following task shown in the table below (Fig.1)

Fig.1 Table of a list of our achievement


We have also managed to successfully construct and evaluate the following BioBrick

Sensory rhodopsin


BBa_K769003
Pconst.(High)-RBS-NpSRII-9a.a.linker_NpHtrII-EnvZ-Double terminator-PompR-RBS-GFP-Double terminator

Tokyo-NoKoGen 2010 created a chimeric sensory rhodopsin with a chemotaxis transducer tar from E. coli (BBa_K225001). We created a blue light sensor, a chimeric sensor rhodopsin, the same sensor domain of rodopsin from Natronobacterium pharaonis (N. pharaonis), and tested to see if the GFP under PompC will show any expression under the light, the light signal received by the sensory rhodopsin. The figure below shows the result. As you can see, we have found that contrary to what we expected, the blue light sensor responded to light by turning off the expression of GFP, and turns on GFP expression in the dark.


The lux operon



Left:BBa_K769022
Middle:BBa_K769011
Right:BBa_K769020


BBa_K769011
Promoter(Pbad)-RBS-lux operon-DT

we have cloned the lux operon Photobacterium phosphoreum and expressed it under arabinose inducible promoter, PBad. We have expressed it inside E. coli and evaluated the luminescence at different temperature, at 30 and 20 degrees celcius. As you can see from the graph below, the strains grown under 20 degrees celcius showed a 5 times higher luminescence intensity than those grown under 30 degrees.


BBa_K769020
lux operon(K769011) + lumP(K769019)

To obtain luminescence of a different color, we have aimed to use a protein called lumazine (lumP) to shift the wavelength peak of the lux luminescence more towards blue light. As you can see in the figure, the wavelength shifted from 487 to 478 nm.


BBa_K769022
lux operon(K769011) + GFP(I13522)

To obtain another color of a luminescence, we have aimed to use the phenomenon BRET, expressing the GFP together with lux operon, so the luminescence of the lux operon will excite the GFP. As a result, we obtained a wavelength shift in the construct expressing the two together.

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